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The Caenorhabditis elegans rop-1 gene encodes the homologue of the human 60-kDa Ro autoantigen
Gene, 167 (1995)227-231 © 1995ElsevierScienceB.V.All rightsreserved.0378-I119/95/509.50
227
GENE 09399
The C~,enorhabditiselegans rop-1 gene encodes the homologue of the huma n 60-kDa Ro autoantigen (Epitopes; nematode; ribonucleoprotein; RNA-binding; SS-A; systemic lupus erythematosus)
Jean-Claude Labb6, Mehrdad Jannatipour and Luis A. Rokeach D~partement de biochimie. Unirersit~de Montrf'al. ltfontr~al, Qw2hecH3C 3J7, Canada
Received by J.K.C. Knowles:19May 1995;Revised/Accepted:2August/16August
995:Receivedat publishers:28 September1995
SUMMARY As a first step toward establishing a genetic system for the elucidation of the cellular role(s) of the Ro ribonucleoproteins (RoRNP), we have cloned the gene encoding the homologue of the human 60-kDa Ro protein (RG60) in Caenorhabdifis elegans (Ce). This Ce gene is present as a single copy and contains a 643-codon open reading frame interrupted by three introns. The encoded protein, Roplp, shares 40% identity and 63% overall similari:y with both the human and amphibian Ro60. Recombinant protein has been produced in Escherichia colt and used to elicit anti-Roplp antibodies. Immunological analysis indicated that the Ro60 epitopes have been poorly conserved. Gene-fusion expression studies in transgenic nematodes will provide a new avenue of cesearch to shed light on the function of these particles.
INTRODUCTION The Ro ribonucleoproteins (RoRNP) are frequently targeted by antibodies (Ab) of patients with the autoimmune diseases systemic lupus erythematosus (SLE) and Sj~gren's syndrome (SS; Tan, 1993). Since the discovery of Ro as an autoantigen about 25 years ago (Clark et al., 1969; Alspaugh and Tan, 1975), the RoRNP have been the subject of intense immunological, biochemical a.u-"~:nolecular characterization ~reviewed in Van Venrooij Correspondence to: Dr. L.A. Rokeach, D/:partcmcnt de biochimie,
Universit6de Montreal,C.P. 6128.succ.Centre-ville. Montrdal.Qu6bec H3C 3J7, Canada. Tel. (1-514) 343.6324; Fax (1-514) 343-6069; e-mail: rokeach~bch.umontreal.ca Abbreviations: aa. amino acid(s); Ab, antibody(ies);bp, base pair(s); Ce. Caenorhabditis elegans: kb. kilobasels)or 1000bp; MBP. maltosebinding protein; NLS, nuclear localization signal(s);nt, nacleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gelelcctrophoresis; PMSF, phenylmethylsulfonylfluoride: RNP, ribonucleoprotein(s);Ro. auto-antigen; Ro60, 60-kDa component of RoRNP; top.I, Ce gene encoding the homologueof Ro60; Roplp, proteinencoded by rop-1; SDS.sodiumdodeeyl sulfate:SS-A,SS antigen A; SLE, systemiclupus erytbematosus; SS, Sj6gren syndrome, TrpE, antbranilate synthase; UTR, untranslated region(s);Y, Ro RNA. SSDI 0378-1119(95)00695-8
et al., 1993; Chan and Buyo~, 1994). Nevertheless, the cellular function of the RoRNP remains elusive. Each RoRNP particle is composed of a single small RNA and at least one proteit~. The Ro RNA moieties are designated Y RNAs. The Y RNAs are characterized by a long stem-loop slructure, whose stem is formed by the 5' and 3' ends of the RNA except for a short singlestranded poly(U) tail at the 3' extremity ~Wolin and Steitz, 1984; Slobbe et al., 1991; O'Brien et al., 1993; Van Gelder et al., 1994). The wain protein constituent associated with the Y RNAs in the RoRNP particles is the 60-kDa Ro autoantigen (Ro60). Cloning and sequencing of cDNAs encoding Ro60 from human nucleated cells IDeutscher et al., 1988; Ben Chetrit et al., 1989) have revealed that this protein contains the RNA-binding domain, designated 'RNP motif', whieh is present in several RNA-binding proteins (reviewed in Burd and Dreyfuss, 1994). In the RoRNP, Ro60 binds directly to the base of the stem of the Y RNAs, and requires a wellconserved bulging C residue in this structure (Wolin and Steitz, 1984; Pruijn et al., 1991; O'Brien et al., 1993). In a recent publication, O'Brien and Wolin (1994) presented the very interesting observation that Xenopus or
228 human Ro60 associate in stable complexes with defective 5S rRNA precursors. Based on these results, these authors proposed that Ror0 could participate in a quality control or discard pathway for 5S rRNA production. With the ultimate aim of developing a genetic system to further characterize the cellular role of the RoRNP, we set out to clone the genes encoding the Ro components in the nematode Caenorhabditis elegans ice). The availability of gene disruption (Zwaal et al., 1993) and germline transformation (Fire and Waterson, 1989; Mello et ai., 1991 ) techniques along with the wealth of genetic and sequence data already gathered, make of Ce an ideal model for our studies. In addition, the use of Ce will allow us to explore the function of the RoRNP both at the cellular and t, rganismal levels. Here we report the cloning and sequencing of the gene encoding the Ce homologue of Ro60. Furthermore, we describe the immunological characterization of this nematodal Ro protein.
EXPERIMENTAL AND DISCUSSION
(a) Ce prodnces a homologue of human Ro60 A cDNA clone, designated cml lg4 (Waterston et al., 1992), potentially encoding a homologue of Ro60 was obtained from Dr. Alan Coulson (Sanger Center, Hinxton, UK). Complete sequencing of t h e c m l l g 4 eDNA revealed that it contains a 643-codon ORF encoding a 72.8-kDa polypeptide (Fig. IA). Searches in the non-redundant (nr) NCBI database showed that the cml lg4 encoded polypeptide shares 40% identity with the Ro60 proteins of human and Xenopus (data not shown and Van Horn et al., 1995). Considering that the La protein from the fruit fly is 33% identical to its human homologue (Bai et al., 1994; Yoo and Wolin, 1994), the degree of conservation between the nematodal protein and Ro60 from vertebrates is remarkable. The similarity between the three polypeptides extends to 63% when conservative changes are taken into account. Moreover, the cm I ! g4 encoded polypeptide also possesses the RNAbinding domain designated 'RNP motif' (reviewed in Burd and Dreyfuss, 1994), which is composed of the two consensus sequences RNPI and RNP2 (Fig. IA). The potential Zn-linger described for human Ro60 (Deutscher et aL, 1988; BenChetrit et al., 1989) is not present in the nematodal protein, Since this latter feature is also absent in the amphibian Ror0 (O'Brien et al., ! 993), this putative motif might not be of significance. Alternatively, the predicted Zn-finger and its function could have arisen at later stage during evolution. Because of the high degree of sequence similarity and the conservation of structural features we concluded that the c m l l g 4 eDNA encodes the Ce homologue of Ro60, and we designated this poly-
peptide Roplp for Ro 12rotein. The evolutionary conservation of Ror0 strongly suggests that it performs an important cellular function(s). During the preparation of this manuscript, Van Horn et al. (1995) reported the cloning of a eDNA encoding Ro60, as well as its associated Y RNA in the nematode Ce. Since the Ro eDNA clone reported by these authors is ctnl lg4, the aa sequence of the encoded polypeptide is identical to the one shown in Fig. lB. (b) Cloning and sequence analysis of the rop-I genc As a prelude to the cloning of the gene encoding Roplp, Southern blotting was carried out using the cm I lg4 eDNA as a probe. A simple pattern of hybridization was obtained (not shown), indicating that the Roplp encoding gene, here designated rop-l, is present as a single copy per genome. Cloning of the rop-I gene was greatly facilitated as the cml Ig4 eDNA had been mapped to chromosome V between the physical markers msp-72ps and col-t2, by The C. elegans Genome Consortium (Sulston et al., 1992). Six genomic cosmids (kindly provided by Dr. Alan Coulson) mapped to this region were analyzed by Southern blotting using the cm I 1g4 cDNA as a probe. One of these cosmids, designated C47A8, produced positive signals (data not shown), and subsequently was used to clone top-1. The nt sequence of this gene is presented in Fig. 1B. The rop-I coding region is interrupted by three introns of 46, 98 and 56 bp at positions 73, 1033 and 1780, respectively (Fig. 1A,B). These are typical Ce introps in terms of their consensus splice sites and their short length (Fields, 1990). The promoter region contains three potential TATA boxes which may be involved in the initiation of top-1 transcription. Also found in this region is a sequence resembling the octamer motif which is involved in the binding of the general transcription factors Oct-I and Oct-2. The 3"-UTR of the mRNA encoded by rop-I displays the sequence AATAAA, the consensus polyadenylation signal. Moreover, this region presents two nt stretches with similarity to the A + U-rich motifs which have been shown to participate in the control of mRNA stability (Sachs, 1993).
(c) Characterization of Roplp To begin its characterization, Rop 1p was overproduced in E, colt as two different fusion proteins. In one case, Roplp coding sequences were cloned in frame with the E. colt trpE gene (encoding anthranilate synthase). The resulting TrpE-Roplp recombinant fusion was purified and used to immunize rabbits for the production of antiRoplp Ab. The sera thereby obtained were then assayed for reactivity against Roplp using the second construction, a fusion with the maltose binding protein, desig-
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Fig. 1. The structure and sequence oftbe rop-I Eerie.(A) Schematic representation of the rop-I Eerie.The promoter region is shown as a shaded box. Exons and introns are denoted as white and black boxes, respectively. The ATG start eodon is marked as a right arrow. The Y-UTR of the mRNA is shown as a dashed box. The positions of relevant restriction enzyme sites are indicated by arrowheads. E, EcoR1; H, Hindlll. (B) The nt sequence of the Ce rop-1 gene and deduced aa sequence. Phage ~. clone cm I Ig4 (Waterston et al, 1992) was kindly provided by Dr. Alan Coulson (The Sanger Center, Hinxton. UK) and recovered as a pBS plasmid (pBSeml Ig4) by in vivo excision as described IStratagene's instructions manual). The nt ~equence of its eDNA was completed by primer walking using the dideoxy chain termination technique. Also provided by Dr. Coulson were six cosmids (B0024. C31D9. C47A8. F07A7. K06BS. ZC86) mapped to chromosome V, between physical markers msp-72ps and col-12, where cml Ig4 wus mapped by The C. elegans Genome Consortium. The cmllg4 eDNA was used as a probe to map the cosmids by Southern blot analysis in order to identify the rop-I sequences. Two HindllI ( 1.9 kb and 1 kb) fragments and one Hindlll-Dra110.6 kb) fragment of cosmid C47A8 produced positive signals and were subsequently isolated, cloned in the pBS vector IStratagene) and the resulting constructions transformed into the E. coil strain XLI-Blue MRF';. Putative TATA box sequences are shown as solid boxes. The potential oetamer motif is denoted by a thick underline. The three introns are delimited by solid, downward triangles. The "RNP motif" is contained within an open box. and the RNPI and RNP2 motifs are shown as shaded boxes at aa 140-145 and 189--197, respectively. Three putative NLS arc shown within open boxes. The polyadenylation signal AATAAA is shown in bold within an open box. Potential A-I-U-rich motifs for R N A degradation m e denoted with a thin underline. The nt sequence o f the em I I g 4 e D N A extends (without the intron sequences) from nt - 9 t o + 2 2 0 7 . Genomic sequences extend from nt - 8 7 6 to + 2 1 4 5 . The nt sequence has GenBank accession N o . U 2 1 4 8 7 .
nated M B P - R o p l p . As shown for one of these sera in Fig. 2A (lanes 1 and 2), the Ab elicited against TrpER o p l p reacted very efficiently with M B P - R o p l p on immunoblots, demonstrating that the antisera produced do contain Ab against R o p l p . When the a n t i - R o p l p sera were assayed with a C e protein extract, they decorated a band of about 65 k D a , even though the predicted molecular mass o f R o p l p is 72.8 k D a (see Fig. 2A, lane 4). This was consistent with the apparent molecular mass o f
R o p l p observed on S D S - P A G E when this polypcptide was synthesized by in vitro transcription/translation. When tested on immunohlots, none o f the a n t i - R o p l p sera reacted with a HeLa extract containing human Ro60 (for example, see Fig. 2A, lane 3). Similarly, the antig o p l p sera did not recognize human Ro60 produced by in vitro transcription/translation when tested in i m m u n o precipitation (Fig. 2B, lane 3), while these Ab readily recognized the in vitro synthesized R o p l p (Fig. 3A, lane
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Fig. 2. lmmunohlot analysis of Roplp. PCR was used to isolate the coding region of Roplp as an expression cassette, having a BamHI restriction site at its 5' terminus. For the production of recombinant Roplp, two expressiml systems were used. The first expression construct was made by inserting the Roplp coding region in frame with the E. coil trpE gene at the BamHI-Sall sites of vector pATH3. In the second construct, the Ropl p coding region was inserted in frame at the BamHISail sites of vector pMAL I New England Biolabs).The resulting expression plasmids, designated pCeRo4089 and pCeRo409L respectively. were transformed in the E. coil strain AP401. Induction of expression from these plasmids and extraction of protein were carried out as described (Rokeach et al., 1991a1.Both induced fusion proteins were clearly visible as a band of about 100 kDa, on 0.1% SDS-10% PAGE stained with Coomassie blue. found in the 6 M urea soluble fractions. The proteins were further purified by cutting the bands out of a preparative 0.1% SDS-10% PA gel. TrpE-Roplp was used to immunize rabbits in order to produce Ab. The cross-reactivity of of these anti Roplp Ab along with different sera against human Ro60 was assayed by Western blot as described previously (Rokeach et al., 1991a)and bound Ab were revealed using the Amersham ECL detection system. Each panel corresponds to a different serum. (A) Rabbit anti-Roplp and (B) pre-immune serum from the same rabbit. IC) rabbit anti-bovine Ro60. Various patients" sera with anti-human RofiOa,loantibod!"es were a~ye~; only representative results are shown: ID) anti-Ro60/anti-Ro52: IE} antiRo60/anti-Ro5.2; IF) anti-Ro60/anti-La; IG} anti-Ro60/anti-La; (H) anti-Ro60/anti-Ro52; ( ! }Normal human serum. The amount of protein present in each lane is as follow: lanes I, 500 ng of TrpE-Roplp; lanes 500 ng of MBP-Roplp; lanes 3, 125 pg of HeLa extract; lanes .4, 250 lag of Ce extract. The black arrow-head marks the position of human Ro60. Lanes M denote protein size markers. 3). Likewise, a n t i - R o A b of a u t o i m m u n e patients a n d a a n t i - b o v i n e R o 6 0 s e r u m did n o t react o n Western blots with E. coli p r o d u c e d r e c o m b i n a n t R o p l p (see Fig. 2, panels C - H ~ lanes I a n d 2). W h e n tested in i m m u n o p r e c i -
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Fig. 3. Immunoprecipitation of in vitro translated products. In vitro translated products, 2.5 pl, were immunoprecipitated with different Ab. Immune complexes were recovered with protein A-Sepharose beads. and analyzed on 0.1% SDS-IO% PAGE followed by fluorugraphy (Amplify.Amersham). (A) Roplp and i BI human Ro60 were synthesized in vitro and immunoprecipitate with different sera. Lanes: i, crude in vitro translation products; 2, serum from rabbit before immunization with TrpE-Rop.lp; 3, rabbit anti-Roplp serum; 4, rabbit anti-bovine Ro60; 5, mAb anti-Ro60: 6-14, human autoimmune sera: 6, anti-Ro60: 7, anti-Ro60/anti-Ro52: 8, anti-Ro60/anti-Ro52; 9, anti-Ro60/anti-La; I0, anti-Ro60/anti-La: IL anti-Ro60/anti-Ro52/anri-La: I2. antiRo60/anti-Ro52; 13, anti-Ro60; 14, anti-Ro60/anti-La: 15. normal human serum. Lanes M represent protein size markers. pitation, the rabbit a n t i - b o v i n e R o 6 0 a n d t w o of the nine h u m a n sera assayed reacted very weakly with the in vitro translated R o p l p (Fig. 3A, lanes 4, 6 a n d 12). These results s h o w t h a t the i m m u n o r e a e t i v e Ro60 epitopes have been p o o r l y conserved. Moreover, R o p l p c a n n o t assemble in vitro with h u m a n Y R N A s , suggesting t h a t the R N A - b i n d i n g d o m a i n of the n e m a t o d a l Ro60 differs f r o m the h u m a n o n e ( d a t a not s h o w n a n d Van H o r n et al., 1995). Because of the a a c o n s e r v a t i o n t h r o u g h o u t the length of the three Ro60 proteins o n e m a y expect t h a t the tertiary s t r u c t u r e of the protein is largely conserved. Therefore, the inability R o p l p to bind to h u m a n Y R N A a n d the lack of recognition of this polypeptide by A b directed to h o m o l o g o u s proteins m a y result from c h a n g e s in a a t h a t are essential for these recognitions.
ACKNOWLEDGEMENTS We apologize for not citing m a n y o t h e r i m p o r t a n t papers d u e to space limitations. We wish to express o u r g r a t i t u d e to Dr. Alan C o u l s o n a n d T h e C. eleeans G e n o m e C o n s o r t i u m for the gift of the e m l l g 4 e D N A a n d the six eosmids. We are grateful to Drs. Gilles Boire a n d E d w a r d C h a n for p r o v i d i n g s o m e of the sera used in this w o r k , a n d J o e Culotti for the N 2 strain. We t h a n k Drs. R o b e r t J. Cedergren, Peter Chidiac, " t r y Hebert a n d Tim Littlejohn for critical reading of the m a n u s c r i p t . We also wish to t h a n k H u g u e s Beaulieu, N a t h a l i e I~thier a n d Steve Titolo for assistance a n d helpful advice, Ms. B. L a p i n s k y for p r e p a r a t i o n of Ab, M. T e m b o for the
231 typing of the manuscript and Yves Villeneuve for the photography
w o r k . L A . R . is a s c h o l a r o f T h e M e d i c a l
Research Council of Canada. This work was supported by
funds
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U n i v e r s i t 6 d e M o n t r 6 a l , to L.A.R.
REFERENCES Alspaugh, M.A. and Tan. E.M.: Antibodies to cellular antigens in Sjrgren's syndrome. J. Clin. Invest. 55 (1975) 1067-1073. Bai, C., Li. Z. and Tolias, P.P.: Developmental characterization of a Drosophila RNA-binding protein homologous to the human systemic lupus erythematosus-associated La/SS-B autoantigen. Mol. Cell. Biol. 14 (1994) 5123-5129. Ben Chetrit, E., Gandy. B.J., Tan, E.M. and Su!livan K.F.: L-,olation and characterization of a cDNA clone encoding the 60.kD component of the human SS-A/Ro ribonucleoprotein autoantigen. J. Clin. Invest. 83 (1989) 1284-1292. Burd, C.G. and Dreyfuss, G.: Conserved structures and diversity of functions of RNA-binding proteins. Science 265 (1994) 615-621. Chan, E.K.L. and Buyon, J.P.: The SS-A/Ro antigen. In: Van Venroij, W.J. and Maini, R.N. (Eds.), Manual of Biological Markers of Disease. Kluwer. Dordmcht. 1994, section 114.1, pp. I 18. Clark, G., Reichlin, M. and Thomas, T.R: Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic erythematosus. J. ImmunoL 102 (1969) 117-122. Fields. C.: Information content of Caenorhabditis elegans splice site sequences varies with intron length. Nucleic Acids Res. 18 11990) 1509-1512. Fire. A. and Waterson. R.H.: Proper expression of myosin genes in tcansgenic nematodes. EMBO J. 8 (1989) 3419-3428. Mello. C.C., Kramer, J.M.. Stinchcomb, D. and Ambros, V.: Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. IO i1991) 3959-3970. O'Brien, C.A.. Margclot, K. and Wolin. S.L.: Xenopns Ro ribonucleoproteins: members of an evolutionarily conserved class of cytoplasmic ribonueleoprnteins. Proc. Natl. Acad. SeL USA 90 (1993) 7250-7254. O'Brien, C.A. and Wolin. S.L.: A possible role for the 6O-kD Ro autoan-
tigen in a discard pathway for defective 5S rRNA precursors. Genes Dev. 8 (1994) 2891-2903. Prnijn, G.J.M,, $1obbe. R.L. and Van Venrooij, WJ.: Analysis of protein-RNA interaction within Ro ribonucleoprotein complexes. Nucleic Acids Res. 19 (1991) 5173-5180. Rokeach, L.A., Haselby, J.A. and Hoch, S.O.: High-level bacterial expression, purification and characterization of human calreticulin. Protein Eng. 4 (1991) 981-987. Sachs, A.B.: Messenger RNA degradation in eukaryotes. Cell 74 (1993) 413-421. Slobbe, R.L.. Pruijn, G.J.M. and Van Venrnoij, WJ.: Ro(SS-A) and La (SS-B) ribonucleoprotein complexes: structure, function and antigenicity. Ann. Med. Intern. 142 ( 1991 ) 592-600. Sulston. J., Du, Z., Thomas, K., Wilson, R., Hillier, L., Staden, R., Halloran, N.. Green, P., Thierry-Mieg, J., Qiu, L. et aL: The C. elegaus genome sequencing project: a beginning. Nature 356 (1992) 37-41. Tan, E.M.: Molecular biology of nuclear autoantigens. Adv. NephroL 22 (1993) 213-236. Van Gelder, C.W.G., Thijsscn. J.P.H.M.. Klassen, E.CJ., Sturchler, C., KroL A.. Van Venrooij, WJ. and Pruijn, GJ.M.: Common structural features of the Ro RNP associated hYI and hY5 RNAs. Nucleic Acids Res. 22 (199412498-2506. Van Horn, D.J., Eisenberg. D., O'Brien, C.A. and Wolin, S.L.: Caeuorhahditis elegaus embryos contain only one major species of Ro RNP. RNA 1 (1995)293-303. Van Venrooij, W.J., Slobbe, R.L. and Pruijn, G.J.M.: Structure and function of La and Ro RNPs. Mol. Biol. Rep. 18 (1993) 113-t 19. Waterston. R., Martin, C., Craxton, M., Huynh. C., Coulson, A., Hillier, L., Durbin, R.. Green, P., Sbownkeen. R., Halloran, N. et aL: A survey of expressed genes in Caenorhabditis elegans. Nature Genet. I (1992) 114-123. Wolin, S.L. and Steitz, LA.: The Ro small cytoplasmic ribonucleoproteins: Identification of the antigenic protein and its binding site on the Ro RNAs. Proc. Natl. Acad. Sci. USA 81 {1984) 1996-2000. Wood, W.B.: The Nematode Caeuorhabditis elegans. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. 1988. Yoo. C.L and Wolin, S.L: La proteins from Drosophila melanogaster and Sacehuramycex cererisiae: A yeast homolog of the La autoantigen is dispensable for growth. Mol. Cell. Biol. 14 (1994) 5412-5424. Zwaal, R.F., Brneks. A.. Van Meurs, J., Groenen, 3.T.M. and Plaste~L R.H.A.: Target-selected gene inactivation in Cae,orhabditis elegaus by using a frozen transposon insertion mutant bank. Proc. Natl. Acad. Sci. USA 90 (1993) 7431 7435.
227
GENE 09399
The C~,enorhabditiselegans rop-1 gene encodes the homologue of the huma n 60-kDa Ro autoantigen (Epitopes; nematode; ribonucleoprotein; RNA-binding; SS-A; systemic lupus erythematosus)
Jean-Claude Labb6, Mehrdad Jannatipour and Luis A. Rokeach D~partement de biochimie. Unirersit~de Montrf'al. ltfontr~al, Qw2hecH3C 3J7, Canada
Received by J.K.C. Knowles:19May 1995;Revised/Accepted:2August/16August
995:Receivedat publishers:28 September1995
SUMMARY As a first step toward establishing a genetic system for the elucidation of the cellular role(s) of the Ro ribonucleoproteins (RoRNP), we have cloned the gene encoding the homologue of the human 60-kDa Ro protein (RG60) in Caenorhabdifis elegans (Ce). This Ce gene is present as a single copy and contains a 643-codon open reading frame interrupted by three introns. The encoded protein, Roplp, shares 40% identity and 63% overall similari:y with both the human and amphibian Ro60. Recombinant protein has been produced in Escherichia colt and used to elicit anti-Roplp antibodies. Immunological analysis indicated that the Ro60 epitopes have been poorly conserved. Gene-fusion expression studies in transgenic nematodes will provide a new avenue of cesearch to shed light on the function of these particles.
INTRODUCTION The Ro ribonucleoproteins (RoRNP) are frequently targeted by antibodies (Ab) of patients with the autoimmune diseases systemic lupus erythematosus (SLE) and Sj~gren's syndrome (SS; Tan, 1993). Since the discovery of Ro as an autoantigen about 25 years ago (Clark et al., 1969; Alspaugh and Tan, 1975), the RoRNP have been the subject of intense immunological, biochemical a.u-"~:nolecular characterization ~reviewed in Van Venrooij Correspondence to: Dr. L.A. Rokeach, D/:partcmcnt de biochimie,
Universit6de Montreal,C.P. 6128.succ.Centre-ville. Montrdal.Qu6bec H3C 3J7, Canada. Tel. (1-514) 343.6324; Fax (1-514) 343-6069; e-mail: rokeach~bch.umontreal.ca Abbreviations: aa. amino acid(s); Ab, antibody(ies);bp, base pair(s); Ce. Caenorhabditis elegans: kb. kilobasels)or 1000bp; MBP. maltosebinding protein; NLS, nuclear localization signal(s);nt, nacleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gelelcctrophoresis; PMSF, phenylmethylsulfonylfluoride: RNP, ribonucleoprotein(s);Ro. auto-antigen; Ro60, 60-kDa component of RoRNP; top.I, Ce gene encoding the homologueof Ro60; Roplp, proteinencoded by rop-1; SDS.sodiumdodeeyl sulfate:SS-A,SS antigen A; SLE, systemiclupus erytbematosus; SS, Sj6gren syndrome, TrpE, antbranilate synthase; UTR, untranslated region(s);Y, Ro RNA. SSDI 0378-1119(95)00695-8
et al., 1993; Chan and Buyo~, 1994). Nevertheless, the cellular function of the RoRNP remains elusive. Each RoRNP particle is composed of a single small RNA and at least one proteit~. The Ro RNA moieties are designated Y RNAs. The Y RNAs are characterized by a long stem-loop slructure, whose stem is formed by the 5' and 3' ends of the RNA except for a short singlestranded poly(U) tail at the 3' extremity ~Wolin and Steitz, 1984; Slobbe et al., 1991; O'Brien et al., 1993; Van Gelder et al., 1994). The wain protein constituent associated with the Y RNAs in the RoRNP particles is the 60-kDa Ro autoantigen (Ro60). Cloning and sequencing of cDNAs encoding Ro60 from human nucleated cells IDeutscher et al., 1988; Ben Chetrit et al., 1989) have revealed that this protein contains the RNA-binding domain, designated 'RNP motif', whieh is present in several RNA-binding proteins (reviewed in Burd and Dreyfuss, 1994). In the RoRNP, Ro60 binds directly to the base of the stem of the Y RNAs, and requires a wellconserved bulging C residue in this structure (Wolin and Steitz, 1984; Pruijn et al., 1991; O'Brien et al., 1993). In a recent publication, O'Brien and Wolin (1994) presented the very interesting observation that Xenopus or
228 human Ro60 associate in stable complexes with defective 5S rRNA precursors. Based on these results, these authors proposed that Ror0 could participate in a quality control or discard pathway for 5S rRNA production. With the ultimate aim of developing a genetic system to further characterize the cellular role of the RoRNP, we set out to clone the genes encoding the Ro components in the nematode Caenorhabditis elegans ice). The availability of gene disruption (Zwaal et al., 1993) and germline transformation (Fire and Waterson, 1989; Mello et ai., 1991 ) techniques along with the wealth of genetic and sequence data already gathered, make of Ce an ideal model for our studies. In addition, the use of Ce will allow us to explore the function of the RoRNP both at the cellular and t, rganismal levels. Here we report the cloning and sequencing of the gene encoding the Ce homologue of Ro60. Furthermore, we describe the immunological characterization of this nematodal Ro protein.
EXPERIMENTAL AND DISCUSSION
(a) Ce prodnces a homologue of human Ro60 A cDNA clone, designated cml lg4 (Waterston et al., 1992), potentially encoding a homologue of Ro60 was obtained from Dr. Alan Coulson (Sanger Center, Hinxton, UK). Complete sequencing of t h e c m l l g 4 eDNA revealed that it contains a 643-codon ORF encoding a 72.8-kDa polypeptide (Fig. IA). Searches in the non-redundant (nr) NCBI database showed that the cml lg4 encoded polypeptide shares 40% identity with the Ro60 proteins of human and Xenopus (data not shown and Van Horn et al., 1995). Considering that the La protein from the fruit fly is 33% identical to its human homologue (Bai et al., 1994; Yoo and Wolin, 1994), the degree of conservation between the nematodal protein and Ro60 from vertebrates is remarkable. The similarity between the three polypeptides extends to 63% when conservative changes are taken into account. Moreover, the cm I ! g4 encoded polypeptide also possesses the RNAbinding domain designated 'RNP motif' (reviewed in Burd and Dreyfuss, 1994), which is composed of the two consensus sequences RNPI and RNP2 (Fig. IA). The potential Zn-linger described for human Ro60 (Deutscher et aL, 1988; BenChetrit et al., 1989) is not present in the nematodal protein, Since this latter feature is also absent in the amphibian Ror0 (O'Brien et al., ! 993), this putative motif might not be of significance. Alternatively, the predicted Zn-finger and its function could have arisen at later stage during evolution. Because of the high degree of sequence similarity and the conservation of structural features we concluded that the c m l l g 4 eDNA encodes the Ce homologue of Ro60, and we designated this poly-
peptide Roplp for Ro 12rotein. The evolutionary conservation of Ror0 strongly suggests that it performs an important cellular function(s). During the preparation of this manuscript, Van Horn et al. (1995) reported the cloning of a eDNA encoding Ro60, as well as its associated Y RNA in the nematode Ce. Since the Ro eDNA clone reported by these authors is ctnl lg4, the aa sequence of the encoded polypeptide is identical to the one shown in Fig. lB. (b) Cloning and sequence analysis of the rop-I genc As a prelude to the cloning of the gene encoding Roplp, Southern blotting was carried out using the cm I lg4 eDNA as a probe. A simple pattern of hybridization was obtained (not shown), indicating that the Roplp encoding gene, here designated rop-l, is present as a single copy per genome. Cloning of the rop-I gene was greatly facilitated as the cml Ig4 eDNA had been mapped to chromosome V between the physical markers msp-72ps and col-t2, by The C. elegans Genome Consortium (Sulston et al., 1992). Six genomic cosmids (kindly provided by Dr. Alan Coulson) mapped to this region were analyzed by Southern blotting using the cm I 1g4 cDNA as a probe. One of these cosmids, designated C47A8, produced positive signals (data not shown), and subsequently was used to clone top-1. The nt sequence of this gene is presented in Fig. 1B. The rop-I coding region is interrupted by three introns of 46, 98 and 56 bp at positions 73, 1033 and 1780, respectively (Fig. 1A,B). These are typical Ce introps in terms of their consensus splice sites and their short length (Fields, 1990). The promoter region contains three potential TATA boxes which may be involved in the initiation of top-1 transcription. Also found in this region is a sequence resembling the octamer motif which is involved in the binding of the general transcription factors Oct-I and Oct-2. The 3"-UTR of the mRNA encoded by rop-I displays the sequence AATAAA, the consensus polyadenylation signal. Moreover, this region presents two nt stretches with similarity to the A + U-rich motifs which have been shown to participate in the control of mRNA stability (Sachs, 1993).
(c) Characterization of Roplp To begin its characterization, Rop 1p was overproduced in E, colt as two different fusion proteins. In one case, Roplp coding sequences were cloned in frame with the E. colt trpE gene (encoding anthranilate synthase). The resulting TrpE-Roplp recombinant fusion was purified and used to immunize rabbits for the production of antiRoplp Ab. The sera thereby obtained were then assayed for reactivity against Roplp using the second construction, a fusion with the maltose binding protein, desig-
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Fig. 1. The structure and sequence oftbe rop-I Eerie.(A) Schematic representation of the rop-I Eerie.The promoter region is shown as a shaded box. Exons and introns are denoted as white and black boxes, respectively. The ATG start eodon is marked as a right arrow. The Y-UTR of the mRNA is shown as a dashed box. The positions of relevant restriction enzyme sites are indicated by arrowheads. E, EcoR1; H, Hindlll. (B) The nt sequence of the Ce rop-1 gene and deduced aa sequence. Phage ~. clone cm I Ig4 (Waterston et al, 1992) was kindly provided by Dr. Alan Coulson (The Sanger Center, Hinxton. UK) and recovered as a pBS plasmid (pBSeml Ig4) by in vivo excision as described IStratagene's instructions manual). The nt ~equence of its eDNA was completed by primer walking using the dideoxy chain termination technique. Also provided by Dr. Coulson were six cosmids (B0024. C31D9. C47A8. F07A7. K06BS. ZC86) mapped to chromosome V, between physical markers msp-72ps and col-12, where cml Ig4 wus mapped by The C. elegans Genome Consortium. The cmllg4 eDNA was used as a probe to map the cosmids by Southern blot analysis in order to identify the rop-I sequences. Two HindllI ( 1.9 kb and 1 kb) fragments and one Hindlll-Dra110.6 kb) fragment of cosmid C47A8 produced positive signals and were subsequently isolated, cloned in the pBS vector IStratagene) and the resulting constructions transformed into the E. coil strain XLI-Blue MRF';. Putative TATA box sequences are shown as solid boxes. The potential oetamer motif is denoted by a thick underline. The three introns are delimited by solid, downward triangles. The "RNP motif" is contained within an open box. and the RNPI and RNP2 motifs are shown as shaded boxes at aa 140-145 and 189--197, respectively. Three putative NLS arc shown within open boxes. The polyadenylation signal AATAAA is shown in bold within an open box. Potential A-I-U-rich motifs for R N A degradation m e denoted with a thin underline. The nt sequence o f the em I I g 4 e D N A extends (without the intron sequences) from nt - 9 t o + 2 2 0 7 . Genomic sequences extend from nt - 8 7 6 to + 2 1 4 5 . The nt sequence has GenBank accession N o . U 2 1 4 8 7 .
nated M B P - R o p l p . As shown for one of these sera in Fig. 2A (lanes 1 and 2), the Ab elicited against TrpER o p l p reacted very efficiently with M B P - R o p l p on immunoblots, demonstrating that the antisera produced do contain Ab against R o p l p . When the a n t i - R o p l p sera were assayed with a C e protein extract, they decorated a band of about 65 k D a , even though the predicted molecular mass o f R o p l p is 72.8 k D a (see Fig. 2A, lane 4). This was consistent with the apparent molecular mass o f
R o p l p observed on S D S - P A G E when this polypcptide was synthesized by in vitro transcription/translation. When tested on immunohlots, none o f the a n t i - R o p l p sera reacted with a HeLa extract containing human Ro60 (for example, see Fig. 2A, lane 3). Similarly, the antig o p l p sera did not recognize human Ro60 produced by in vitro transcription/translation when tested in i m m u n o precipitation (Fig. 2B, lane 3), while these Ab readily recognized the in vitro synthesized R o p l p (Fig. 3A, lane
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Fig. 3. Immunoprecipitation of in vitro translated products. In vitro translated products, 2.5 pl, were immunoprecipitated with different Ab. Immune complexes were recovered with protein A-Sepharose beads. and analyzed on 0.1% SDS-IO% PAGE followed by fluorugraphy (Amplify.Amersham). (A) Roplp and i BI human Ro60 were synthesized in vitro and immunoprecipitate with different sera. Lanes: i, crude in vitro translation products; 2, serum from rabbit before immunization with TrpE-Rop.lp; 3, rabbit anti-Roplp serum; 4, rabbit anti-bovine Ro60; 5, mAb anti-Ro60: 6-14, human autoimmune sera: 6, anti-Ro60: 7, anti-Ro60/anti-Ro52: 8, anti-Ro60/anti-Ro52; 9, anti-Ro60/anti-La; I0, anti-Ro60/anti-La: IL anti-Ro60/anti-Ro52/anri-La: I2. antiRo60/anti-Ro52; 13, anti-Ro60; 14, anti-Ro60/anti-La: 15. normal human serum. Lanes M represent protein size markers. pitation, the rabbit a n t i - b o v i n e R o 6 0 a n d t w o of the nine h u m a n sera assayed reacted very weakly with the in vitro translated R o p l p (Fig. 3A, lanes 4, 6 a n d 12). These results s h o w t h a t the i m m u n o r e a e t i v e Ro60 epitopes have been p o o r l y conserved. Moreover, R o p l p c a n n o t assemble in vitro with h u m a n Y R N A s , suggesting t h a t the R N A - b i n d i n g d o m a i n of the n e m a t o d a l Ro60 differs f r o m the h u m a n o n e ( d a t a not s h o w n a n d Van H o r n et al., 1995). Because of the a a c o n s e r v a t i o n t h r o u g h o u t the length of the three Ro60 proteins o n e m a y expect t h a t the tertiary s t r u c t u r e of the protein is largely conserved. Therefore, the inability R o p l p to bind to h u m a n Y R N A a n d the lack of recognition of this polypeptide by A b directed to h o m o l o g o u s proteins m a y result from c h a n g e s in a a t h a t are essential for these recognitions.
ACKNOWLEDGEMENTS We apologize for not citing m a n y o t h e r i m p o r t a n t papers d u e to space limitations. We wish to express o u r g r a t i t u d e to Dr. Alan C o u l s o n a n d T h e C. eleeans G e n o m e C o n s o r t i u m for the gift of the e m l l g 4 e D N A a n d the six eosmids. We are grateful to Drs. Gilles Boire a n d E d w a r d C h a n for p r o v i d i n g s o m e of the sera used in this w o r k , a n d J o e Culotti for the N 2 strain. We t h a n k Drs. R o b e r t J. Cedergren, Peter Chidiac, " t r y Hebert a n d Tim Littlejohn for critical reading of the m a n u s c r i p t . We also wish to t h a n k H u g u e s Beaulieu, N a t h a l i e I~thier a n d Steve Titolo for assistance a n d helpful advice, Ms. B. L a p i n s k y for p r e p a r a t i o n of Ab, M. T e m b o for the
231 typing of the manuscript and Yves Villeneuve for the photography
w o r k . L A . R . is a s c h o l a r o f T h e M e d i c a l
Research Council of Canada. This work was supported by
funds
CAFIR,
FIRFM,
Facult6
de
m6decine.
U n i v e r s i t 6 d e M o n t r 6 a l , to L.A.R.
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